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This Week’s Citation Classic ®
CC/NUMBER 51
DECEMBER 17, 1990
Sutcliffe J G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322.
Cold Spring Harbor Symp. 43:77-90, 1979.
[Biological Laboratories, Harvard University, Cambridge, MA]
This paper describes the nucleotide sequence of
pBR322 and the strategy that was developed to ensure
the accuracy of the results. The biological features
encoded by the plasmid are interpreted through the
sequence, as are features that enhance its value as a
cloning vector. [The SCI® indicates that this paper
has been cited in more than 1600 publications, making it the most-cited paper from this journal.[
The Sequence of pBR322
I. Gregor Sutcliffe
Department of Molecular Biology
Research Institute of scripps Clinic
10666 North Torrey Pines Road
La Jolla, CA 92037”
The middle 1970s were an exciting lime to be a
graduate student in molecular biology, especially if
one were fortunate enough to be in the large group
jointly led by Jim Watson, Wally Gilbert (my thesis
adviser), Klaus Weber, and David Dressier at the
Harvard Biological Laboratories. Many important
papers were written during that period by members
of the group, induding one by Gilbert and Allan
Maxam describinga chemical method for determin1
ing DNA sequences.
DNA sequencing technology was crude at that
time, and previous methods were not highly reliable. Indeed, the landmark sequence
of the 4X174
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genome from Sanger’s group using DNA analysis
combined with brute force isolation and sequencing
of all of the 4X1 74 RNA transcripts and protein
products had just been published, conduding that
“errors and uncertainties can only be eliminated by
more laborious experiments and...it would probably
be a long time before the complete sequence could
be established. We are not certain that there is any
scientific justification for establishing every detail....” The Maxam-Gilbert method looked capable
of producing more accurate data, but it had been
applied only to relatively short regions of DNA.
Gilbert and his colleague, Jeremy Knowles, wondered whether the sequence of an entire protein,
3-lactamase, could be obtained solely from structural analysis of its gene. Partial amino acid sequence data, which Knowles kept locked in his
desk, were available from R.P. Ambler and G.K.
Scott in Edinburgh. I was chosen for this test ofthe
Maxam-Gilbert sequencing method in part because
I had lobbied for an assessment of its accuracy. I
selected the 3-lactamase gene of pBR322 for study
because it was evident this was a superior doning
vector and any information about it would be valuable. Over seven months, I learned the method,
developed strategies for studying an entire gene,
and scaled-up the technology, espethlly gel electrophoresis, since then it could require 10 gels to obtain 100 bases (now it’s kilobases/gel). Every base
was critical as errors could alter the apparent triplet
reading frame and lead to an imaginary predicted
protein sequence. Thus I developed careful checking procedures to give me confidence 3in the final
1,100 base 3-lactamase gene sequence. The moment of truth came over tea at Knowles’s house on
a weekend afternoon. We went residue by residue,
comparing my handwritten nudeotide and deduced
amino acid sequence to the Ambler-Scott data. We
found only one discrepancy that turned out to result
from a strain difference rather than error.
The comparison was exhilarating and its result
gratifying given the effort that went into achieving
it. This increased my incentive to complete the entire pBR322 sequence, a project that required more
than 400 gels and was finished six months later. The
final 4,362 base sequence was proofread with the
help of most members of the group. Over the years,
only one error has been found, although an embarrassing one, as it affected the interpretation of the
tetracycline resistance mechanism. Nevertheless,
the magnitude of the project was unprecedented
and certainly changed the perception I, and probably others, had of the power of DNA sequence
analysis.
This and a related paper containing restriction
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cleavage maps derived from the sequence have
been cited because the data they contain have direct application in many studies. pBR322 became
the cloning vector of choice, in part because of the
availability of its sequence. However, the greater
value of the work was that it demonstrated the
startling accuracy and speed with which kilobases
of DNA sequence could be determined. This realization has contributed to the consideration of the
sequence of the human genome as an achievable
goal. That carefully determined sequences provide
solid foundations for experimental work is evident
in most molecular approaches taken in the 1980s.
i. Maxam AM & Gilbert W. A new method for sequencing DNA. Proc. Nat. Arad. Sd. USA 74:560-4, 1977.
(Cited 5.555 times.)
2. Sanger F, Air G M, Barrell B G. Brown N L, Coulson AR, Fiddes J C, Hutchison C A, Slocombe PM & Smith M.
Nucleotide sequence of bacteriophoge ~X 174. Nature 265:687-95. 977. (Cited 880 times.)
3. Sutcliffe J G. Nucleotide sequence of the arnpicillio resistance gene of E. celi plasmid pBR322.
Proc. Nat. Acad. Sd. USA 75:3737-41, 1978. (Cited 405 times.)
4.
pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 436! nocleotide
pairs )ovg. NucL Arid. Rev. 5:2721-8. 978. (Cited 780 times.)
°ReceivedAugust 3. 990
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